Our purpose is to determine the kinetic pathway of folding of a small protein, ribonuclease A (RNaseA), and to deduce the mechanism of folding from the pathway and the structures of the intermediates. We have recently succeeded in finding conditions where folding intermediates accumulate transiently. These conditions are low temperatures (0-10 degrees C) at neutral pH; addition of a stabilizing salt ((NH4)2SO4 or Na2SO4) enhances the accumulation of intermediates. Unfolded RNaseA contains an equilibrium mixture of two classes of molecules: a major (80 percent) slow-folding class (US) and a minor (20 percent) fast-folding class (UF). We study here the folding of the US class. The US-UF reaction in unfolded RNaseA is believed to be proline isomerization and the US molecules are thought to contain "wrong" isomers of one or more proline residues. Our current plans include: (1) further tests of the proline isomerization explanation of the US-UF reaction, (2) study of the 3H-labeled, exchangeable amide protons which are trapped during the folding of US by the formation of folding intermediates, and (3) study of the structure of an equilibrium folding intermediate formed by S-protein (a fragment of RNaseA, containing residues 21-124) at pH 1.7, below 30 degrees C.